It is known from WO-2005/000280 to use the exine coatings of naturally derived (typically plant) spores as delivery vehicles for active substances such as pharmaceuticals and dietetic substances. These coatings can be isolated from spores by successive treatments, for example with organic solvents, alkali and acid so as to remove the lipid, carbohydrate, protein and nucleic acid components that may be attached to or contained within the exine shell. Enzymatic methods have also been used to isolate the exine coating from other components of a spore.
Exine coatings (or shells) take the form of essentially hollow capsules that can be impregnated or filled with, or chemically or physically bound to, another substance. According to WO-2005/000280, a pharmaceutical or dietetic active substance may be physically or chemically bound to, adsorbed on or more typically encapsulated within such a hollow exine shell. The exine/active substance combination may then be formulated—often mixed with conventional excipients, diluents or carriers and/or with release rate modifiers—for the desired mode of delivery, for example oral, buccal or pulmonary delivery.
WO-2007/012857 discloses the use of exine shells as delivery vehicles in topical formulations. This document describes how the exine shells, despite their mechanical and chemical strength, can be caused, by gentle rubbing, to release a substance encapsulated within them. This makes the exine shells particularly suitable for topical delivery of substances, such as cosmetics or sunscreens, to surfaces such as the skin.
Sometimes, when formulating an active substance, it is necessary to protect the substance, at least temporarily, from external influences such as light, moisture or oxygen (air). This may be for the purpose of improving the storage stability of the formulation, or it may be to ensure that the formulation reaches, following its delivery to a patient, the appropriate part of the body.
Exine shells can themselves provide a degree of protection for an encapsulated active substance, for instance from atmospheric effects such as light and/or oxygen (air), and therefore from premature degradation. The physical protection they provide can also help reduce loss of the active substance by evaporation, diffusion or leaching. It has also been found (as disclosed in WO-2007/012856) that in many cases an exine shell can itself act as an antioxidant, rather than merely as a physical barrier to oxygen (air), this effect being observable even when an active substance is outside of, rather than encapsulated within, the shell.
Useful exine coatings may be isolated from spores by successive treatments with organic solvents, alkali and acid so as to remove the lipid, carbohydrate, protein and nucleic acid components that may be attached to or contained within the exine shell. In WO-2007/012856 such exine shells isolated by acid hydrolysis (for example, with phosphoric acid) followed by base hydrolysis (for example, with potassium hydroxide) are designated “AHS”. Also disclosed are exine shells designated “BHS” which were subjected only to base hydrolysis (for example, with potassium hydroxide). The BHS samples comprised not only the exine shell but also a proportion of the cellulosic intine layer normally removed during the acid treatment. It was found that some BHS exine coatings were particularly effective in reducing the oxidation rates of oils encapsulated therein when exposed to UV light.
Exine coatings produced by methods disclosed in the foregoing documents are typically chocolate brown in colour. Such a coloured product can be undesirable for many applications. For example, in the case of cosmetics, food products and certain pharmaceutical applications, dark particles may be visible within a lighter product or may appear to discolour it. Even if it is desired to colour the product, a dark exine delivery vehicle may still be visible or may reduce the attractiveness of the applied colour, for example by making it appear dull or muddy. There can be a need, therefore, to lighten the colour of exine coatings used as delivery vehicles in such applications, and a creamy or relatively pale colour can be highly desirable.
In Svensk Botanisk Tidskrift, 54, 4 1960 by G Erdtman, there is described a method for preparing herbarium material for mounting on optical microscope slides. Powdered flowers, buds or isolated stamens were subjected to acetolysis using a mixture of acetic anhydride and concentrated sulphuric acid at 70° C. This relatively harsh acetolysis procedure for isolating pollen grains in preparation for mounting on microscope slides caused the grains to become so much darker in colour, especially in spores from species such as Geranium and Scabiosa, that they could not be microscopically observed. Therefore, before such observations were carried out, the pollen grains had to be bleached. Bleaching was undertaken using glacial acetic acid, saturated sodium chlorate solution and concentrated hydrochloric acid. However, as shown in Comparative Example 14 below, the end product of this process still has a degree of whiteness insufficient to be suitable for use in food, cosmetics and pharmaceuticals. In addition, the use of Erdtman's method tends to cause fragmentation of the pollen shells, which was acceptable in the context of preparing a sample for microscopic study.
Such fragmentation is not surprising, since sporopollenin is known to be degraded by exposure to oxidising agents (see G. Shaw, “The chemistry of sporopollenin.” in J. Brooks et al. (eds.) Sporopollenin. (1971) London & New York: Academic Press, p. 305-348). Therefore, there is a disincentive to subject exine shells to bleaching in order to lighten their colour, where the shells are intended for use as delivery vehicles (e.g., WO-2005/000280 & WO-2007/012857), for which purpose substantially intact shells are required. As well as being expected to compromise the structural integrity of the resultant shells, bleaching might also be expected to compromise their antioxidant properties such as were described in WO-2007/012856. Oxidative degradation and loss of antioxidant effects could both be detrimental for shells intended to be used as delivery vehicles in for example foods, cosmetics and pharmaceuticals.
Surprisingly, however, the present inventors have found that exine shells prepared under hydrolysis treatment conditions can be whitened to an appropriate degree without undue structural degradation, despite the vulnerability of sporopollenin to oxidative degradation. Such shells have also been found to retain antioxidant activity. The resultant whitened exine shells can be suitable for encapsulating active substances for delivery, especially in foods, cosmetics and pharmaceuticals.